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目的:构建靶向化学趋化因子受体1(CXCR1)的短发夹小干扰RNA(shRNA)质粒表达载体。方法:针对人CXCR1基因的mRNA序列,按RNA干扰靶位点的设计原则,设计并构建靶向CXCR1基因的3个shRNA质粒表达载体和1个阴性对照质粒表达载体,经酶切和测序确认构建成功后,转染胃癌细胞MKN45,RT-PCR和Western blot检测CXCR1 mRNA和蛋白的表达。结果:经酶切和测序证实,3个靶向CXCR1基因的shRNA真核表达质粒均构建成功;与未转染和转染阴性对照质粒的MKN45细胞比较,转染3种shRNA质粒的MKN45细胞,CXCR1 mRNA和蛋白水平均明显下调(均P<0.05)。结论:靶向CXCR1基因的shRNA真核表达质粒的成功构建,为进一步研究CXCR1在胃癌中的功能和实验性靶向治疗提供了初步的基础。
Objective: To construct a short hairpin RNA interference (shRNA) plasmid expression vector targeting chemokine receptor 1 (CXCR1). Methods: According to the design of RNA interference targeting site of human CXCR1 mRNA, three shRNA plasmid expression vectors targeting to CXCR1 gene and one negative control plasmid plasmid were designed and constructed. The constructed plasmid was confirmed by restriction enzyme digestion and sequencing After successful, the expression of CXCR1 mRNA and protein was detected by RT-PCR and Western blot. Results: The three eukaryotic expression plasmids targeting CXCR1 gene were confirmed by restriction enzyme digestion and sequencing. Compared with untransfected and transfected MKN45 cells transfected with negative control plasmid, MKN45 cells transfected with three kinds of shRNA plasmids, CXCR1 mRNA and protein levels were significantly decreased (all P <0.05). Conclusion: The successful construction of shRNA eukaryotic expression plasmid targeting CXCR1 gene provides a preliminary basis for further study on the function and experimental targeted therapy of CXCR1 in gastric cancer.